ABSTRACT: Little is known about the genetic diversity of pale flax (Linum bienne Mill.), the wild progenitor of cultivated flax (L. usitatissimum L.), and ex situ germplasm of pale flax was scarce. Effort was made to collect 34 pale flax accessions and five landrace accessions of cultivated flax in Turkey. The inter simple sequence repeat (ISSR) technique was applied to characterize this set of flax germplasm, along with one Turkish cultivar, one Russian cultivar, five winter and four dehiscent type accessions of cultivated flax. Twenty-four ISSR primer pairs detected a total of 311 DNA fragments, of which 298 bands were polymorphic across 493 flax samples (roughly 10 samples per accession). These polymorphic bands had frequencies ranging from 0.002 to 0.998 and averaging 0.38. Accession-specific ISSR variation (Fst values) ranged from 0.469 to 0.514 and averaged 0.493. There was 49.3% ISSR variation resided among these 50 accessions, 35.9% harbored among landrace, winter, dehiscent types of cultivated flax and pale flax, and 38.2% present among 34 pale flax accessions. Pale flax displayed more ISSR variation than landraces and dehiscent type, but less than winter type, of cultivated flax. Clustering 493 individual plants revealed that these assayed plants were largely grouped according to their plant types and that pale flax was genetically more close to the dehiscent type, followed by the winter type and landrace, of cultivated flax. Pale flax collected within the geographic range of 180 km displayed a significant spatial genetic autocorrelation. Genetic distances among the pale flax accessions were significantly associated with their geographic distances and elevation differences. These findings are significant for understanding flax domestication and its primary gene pool.